PubMed Health⌕ Search

Biomedical subjects

M Conti

Publications and source records attributed to M Conti.

At least 127 records · Page 7Linked to original sources
PubMed1997

Selective stimulation of a cAMP-specific phosphodiesterase (PDE4A5) isoform by phosphatidic acid molecular species endogenously formed in rat thymocytes.

We have previously reported that concanavalin A (ConA) stimulation of rat thymocytes induces an increase in the cellular phosphatidic acid mass as well as a change in its fatty acid composition. An increase in phosphodiesterase (PDE) activity, mostly due to cAMP-specific (PDE4) isoforms, has also been observed in thymocytes stimulated by ConA. Furthermore, phosphatidic acid was able to stimulate PDE4 activity in vitro. In the present study, cAMP levels have been shown to decrease upon ConA stimulation of thymocytes. Decreasing phosphatidic acid level using diacylglycerol kinase inhibitors induced a parallel decrease of the ConA-stimulated cAMP-specific PDE activity in these cells. Analyses of phosphatidic acid molecular species in cells stimulated for 5 min by ConA revealed a significant increase in 1-stearoyl-2-arachidonoyl-sn-glycerol-3-phosphate and a relative decrease in the other molecular species of phosphatidic acid, mainly species containing palmitate. On the other hand, phosphatidic acid extracted from ConA-stimulated cells activated more efficiently the recombinant PDE4A5 isoform in vitro, as compared to phosphatidic acid extracted from unstimulated cells. In addition, phosphatidic acid species containing unsaturated fatty acids were stimulatory, while those containing two saturated fatty acids had only a marginal effect on the enzyme activity. Taken together, these data suggest that the mitogenic stimulation of thymocytes is accompanied by the synthesis of peculiar phosphatidic acid molecular species able to activate a PDE4 isoform. This activation might be of physiological relevance since cAMP is a major negative effector of the mitogenic response.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Study of haptoglobin-hemoglobin complexes by titration curves, capillary electrophoresis and capillary isoelectric focusing.

A novel method is described for monitoring complex formation between macromolecules, based on combined isoelectric focusing-electrophoresis in capillaries. The example studied is the binding of serum haptoglobin (Hp) to hemoglobin (Hb). A known amount of Hb is focused in a capillary in a pH 6-8 range (pI of Hb = 7.0) and thus kept temporarily "immobilized" in the electrophoretic chamber. Subsequently, increasing amounts of ligand (Hp) are loaded cathodically and allowed to sweep past the focused Hb zone. As the complex formed has a pI value well-outside the bounds of such a pH gradient (the 1:1 molar Hb-Hp complex has a pI of 5.5, the 1 to 1/2 molar Hp-Hb complex has a pI of 5.0) it escapes immobilization and moves past the detector window, where it is monitored and quantified. Since the detector is set at 416 nm, where only Hb absorbs, and since the molar extinction coefficient of Hb is well known, it is quite easy to calculate the molar amount of Hb bound to the complex. As an additional check, the amount of unreacted Hb can now be mobilized by disrupting the pH gradient and allowing this residual free Hb to also reach the detector and be quantified. The method is easy, fast, simple and fully automated and thus could represent a valid alternative to existing methods in clinical chemistry for quantifying the amount of Hp in human sera in pathological conditions, such as hemolytic anemias and transfusion reactions.

Ampholyte Mixtures↗
PubMed1997

Abnormal ventricular repolarization mimicking myocardial infarction after heterocyclic antidepressant overdose.

In 2 young adult women who experienced acute heterocyclic antidepressant intoxication, we found a quite unusual electrocardiographic pattern characterized by abnormal ST-tract elevation in the right precordial leads associated with a marked QRS widening (right bundle branch block and left anterior fascicular block type). Because serum electrolyte imbalance and acute myocardial ischemic events were excluded, the mechanism by which antidepressant overdose may produce such elevation of the ST tract remains unclear.

Adult↗
PubMed1997

Cesarean section using the Misgav Ladach method.

OBJECTIVE: To stress the advantages of the Misgav Ladach method for cesarean section. STUDY DESIGN: In this study operative details and the postoperative course of 139 patients who underwent cesarean section according to the Misgav Ladach method in 1995-96 are presented. RESULTS: The Misgav Ladach method reduces operation time, time of child delivery, and time of recovery. The rates of febrile morbidity, wound infection and wound dehiscence are not affected by the new technique. CONCLUSION: Our study highlights the efficiency and safety of the Misgav Ladach method, and points out the speeded recovery, with early ambulation and resumption of drinking and eating, that makes the cesarean section delivery closer and closer to natural childbirth.

Adult↗
PubMed1997

Activation of phosphodiesterase IV during desensitization of the A2A adenosine receptor-mediated cyclic AMP response in rat pheochromocytoma (PC12) cells.

Prolonged activation of an A2A adenosine receptor significantly inhibits the cellular response to subsequent stimulation (A2A desensitization). We have reported previously that activation of phosphodiesterase (PDE) contributes to A2A desensitization in PC12 cells. In the present study, we show that a type IV PDE (PDE4)-selective inhibitor (Ro 20-1724) effectively blocks the increase in PDE activity in desensitized cells. Thus, PDE4 appears to be the PDE specifically activated during A2A desensitization in PC12 cells. Prolonged treatment of PC12 cells with an A2A-selective agonist (CGS21680) leads to increased PDE4 activity in a dose-dependent manner, which can be blocked by an A2A-selective antagonist [8-(3-chlorostyryl)caffeine]. Using two PDE4 antibodies, we were able to demonstrate that the levels of two PDE4-immunoreactive bands (72 and 79 kDa) were increased significantly during A2A desensitization. Prolonged treatment with forskolin to elevate intracellular cyclic AMP contents also resulted in increased PDE4 activity. In addition, activation of PDE4 activity during A2A desensitization could be blocked by a protein kinase A (PKA)-selective inhibitor (H89) and was not observed in a PKA-deficient PC12 cell line (A123). Taken together, activation of PDE4 via a cyclic AMP/PKA-dependent pathway plays a critical role in dampening the signal of the A2A receptor.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Noradrenergic activity differentially regulates the expression of rolipram-sensitive, high-affinity cyclic AMP phosphodiesterase (PDE4) in rat brain.

In a previous study, it was observed that the activity of rolipram-sensitive, low-Km, cyclic AMP phosphodiesterase (PDE4) was decreased in vivo with diminished noradrenergic stimulation. The results of the present experiments indicated that the reduction in the activity may be associated with down-regulation of PDE4 protein. Immunoblot analysis using PDE4-specific, subfamily-nonspecific antibody (K116) revealed four major bands of PDE4 in rat cerebral cortex; those with apparent molecular masses of 109 and 102 kDa are variants of PDE4A. Diminished noradrenergic activity, produced by intracerebroventricular infusion of 6-hydroxydopamine (6-OHDA) or chronic subcutaneous infusion of propranolol, decreased the intensities of the protein bands for the 109- and 102-kDa PDE4A variants in rat cerebral cortex but not of the 98- or 91-kDa PDE4 forms. 6-OHDA-induced noradrenergic lesioning also decreased the content of 102-kDa PDE4A in hippocampus as labeled by PDE4A-specific antibody (C-PDE4A). Enhanced noradrenergic stimulation up-regulated PDE4 in cerebral cortex. This was indicated by the finding that repeated treatment with desipramine increased the intensity of the protein band for the 102-kDa PDE4 but not for the other variants of PDE4. These results suggest that PDE4 subtypes are differentially regulated at the level of expression, as evidenced by an apparent change in the amount of PDE4 protein, following changes in noradrenergic activity. These observations are consistent with the notion that PDE4s, especially the PDE4A variants with molecular masses of 109 and 102 kDa, play an important role in maintaining the homeostasis of the noradrenergic signal transduction system in the brain and may be involved in the mediation of antidepressant activity.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

Telomerase activity in female and male rat germ cells undergoing meiosis and in early embryos.

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA at the ends of eukaryotic chromosomes. It has been hypothesized that telomerase activity is necessary for cellular immortalization and that telomerase activity is present in cells of germline origin. The objective of the present study was to determine the level of telomerase activity in the following rat cells: 1) oocytes from follicles at different stages of development, 2) spermatogenic cells, and 3) early embryos. Telomerase activity was quantitated using a recently developed, sensitive polymerase chain reaction-based assay and a human kidney cell line (293) as a standard. Telomerase activity was found in oocytes from early antral and preovulatory follicles, as well as in ovulated oocytes. The level of enzyme activity in early antral and preovulatory follicles was comparable to that of the 293 cells, while levels in ovulated oocytes were 50-fold lower. Telomerase activity was present in even lower levels in pachytene spermatocytes and round spermatids, and no telomerase activity was detected in spermatozoa from either the caput or the cauda epididymis. After fertilization, telomerase activity was present in 4-cell embryos. Telomerase activity was also detected in several rat somatic tissues. These data demonstrate that telomerase activity is present in germ cells at several stages of differentiation, with the exception of spermatozoa, and suggest that telomerase activity may be important during meiosis. The high levels of telomerase activity in individual oocytes may serve as a marker for monitoring the effects of hormonal agents, aging, and toxins on oocyte quality.

Animals↗
PubMed1997

Selective activation of rolipram-sensitive, cAMP-specific phosphodiesterase isoforms by phosphatidic acid.

In rat thymic lymphocytes, accumulation of phosphatidic acid (PA) occurs at the same time as decrease in cAMP levels and activation of a cAMP-specific phosphodiesterase (PDE) [type 4, EC 3.1.4.17 (PDE4)]. We investigated the nature of the PDE activated by PA and the mechanism of activation by using recombinant cAMP-specific PDE4 isoforms derived from three different genes (PDE4A, PDE4B, and PDE4D). The "long" variants expressed from each gene (PDE4A5, PDE4B1, and PDE4D3) were activated by PA, whereas the "short" variants (PDE4A1, PDE4B2, PDE4D1, and PDE4D2) were not. Phosphatidylserine was an activator that was as effective as PA, whereas phosphatidylcholine was ineffective, indicating that activation was restricted to anionic phospholipids. PA caused an increase in the Vmax value of PDE4D3 without affecting the Km value of the enzyme for the cAMP substrate. PA also caused a change in the Mg2+ requirement for hydrolysis. Half-maximal stimulation of the PDE was obtained with approximately 10 microg/ml PA. Although protein kinase A-mediated phosphorylation of PDE4D3 produces effects similar to those elicited by PA, the mechanism of PA-induced activation was not found to involve a phosphorylation. Instead, several observations suggest that PA may directly interact with the enzyme. The stimulation of cAMP PDEs by PA and other acidic phospholipids may be a mechanism by which growth factors and hormones modulate the cAMP-dependent signal transduction pathway during cell stimulation.

Animals↗
PubMed1997

Characterization of an intronic promoter of a cyclic adenosine 3',5'-monophosphate (cAMP)-specific phosphodiesterase gene that confers hormone and cAMP inducibility.

In the Sertoli cell, FSH stimulates transcription of a cAMP-phosphodiesterase (PDE) gene (PDE4D) and accumulation of corresponding mRNA and PDE protein. The regulation of this PDE gene is an important component of the desensitization state induced by this hormone. Given the ubiquitous nature of this regulation controlling cAMP levels, the molecular basis for the PDE4D induction was further investigated. FSH stimulation of the Sertoli cell causes the accumulation of only two of the four known PDE4D mRNAs (PDE4D1 and PDE4D2). The promoter controlling the expression of these two messages was identified and characterized. An EcoRI fragment containing a coding exon as well as 5'-upstream sequence of the PDE4D1/2 mRNA was isolated from rat genomic libraries and sequenced. No TATA box was identified, but GC-rich regions were present upstream of the putative translation start site. RNAse protection and PCR analysis indicated the presence of at least two distinct cap sites. This genomic region had promoter activity when transfected both in Sertoli and MA-10 cells. Deletion mutation indicated that basal promoter activity was contributed by regions upstream of both cap sites. Transcription from this promoter was activated by FSH and (Bu)2cAMP, and elements responsible for cAMP regulation were present upstream from the second cap site. These data demonstrate that an intronic promoter that is cAMP- and hormone-inducible directs the expression of these truncated PDE proteins.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1997

[Comparative, randomized, parallel clinical study of the effectiveness and safety of aceclofenac vs. paracetamol in the treatment of viral pharyngoamygdalitis].

The purpose of this open, controlled, and randomized study was to evaluate the effectiveness of aceclofenac compared with paracetamol in improving the signs and symptoms of viral pharyngoamygdalitis and to evaluate the safety of both drugs. Thirty outpatients (age range 18-65 years) with acute viral pharyngoamygdalitis received either aceclofenac 100 mg/12 h or paracetamol 650 mg/ 12 h per os. Patients were evaluated at baseline and on days 1, 3, and 7 after beginning treatment. The parameters of effectiveness evaluated were: severity of pharyngoamygdalitis, spontaneous pharyngeal pain, saliva swallowing, and duration of pharyngeal pain. The results showed that the aceclofenac group had a significant decrease in the severity of pharyngoamygdalitis and spontaneous pharyngeal pain after day 1 of treatment (p < 0.001), but the paracetamol group showed no improvement in these parameters until day 3 of treatment (p < 0.001). There were no adverse reactions in either group. Therefore, patients treated with aceclofenac showed an earlier improvement in the signs and symptoms of acute viral pharyngoamygdalitis than those treated with paracetamol.

Acetaminophen↗
PubMed1997

[Accident during retinal fluorescein angiography].

OBJECTIVE: To evaluate adverse reactions frequency following fluorescein i.v. administration to perform retinoic angiography (ARF). DESIGN: Retrospective study. SETTING: Eye Clinic of the University of Florence. MATERIALS AND METHODS: 6524 patients undergoing 10,003 ARF. PARAMETERS EVALUATED: For each patient age, sex, concomitant diseases, previous ARF, allergic history and any adverse reaction were evaluated. RESULTS: Adverse experiences were described in 7.5% of 6524 patients. Most of the observed reactions were mild: nausea (3.8%), vomiting (0.43%), vasovagal phenomena (1.18%), anaphylactoid reactions (1.84%). A major rate of adverse reactions were observed in patients previously submitted to ARF who reported in the anamnesis a previous adverse reaction, and in those with a positive allergic history. Severe reactions occurred in 0.27% of patients, but only in 2 of 6524 patients a rianimatory treatment (0.03) was necessary. CONCLUSION: From the data retrospective analysis it was observed that the manifestation of mild adverse effects was to be expected in many cases. It was therefore possible to point out classes of patients at risk (patients who reported a previous adverse reaction after ARF and patients with positive allergic history). Unfortunately it was not possible to expect and so to prevent the severe effects. Thus it is recommended to perform ARF in an adequately equipped room.

Adolescent↗
PubMed1996

Effects of low-dose heparin infusion on arterial endothelin-1 release in humans.

BACKGROUND: The aim of this study was to evaluate the effect of low-dose heparin infusion on arterialized endothelin-1 (ET-1) release in the presence of fasting or high insulin levels in healthy humans. METHODS AND RESULTS: Eleven normal subjects underwent two tests in random order lasting 240 minutes. A primed (250 IU), continuous heparin (600 IU/h) infusion was performed in test 1; saline was infused in test 2 as control. At 120 minutes, a euglycemic hyperinsulinemic clamp (25 mU.kg-1.h-1) was started that lasted 2 hours in both tests. Two hours after heparin infusion (test 1), ET-1 levels decreased by 32% (3.52 +/- 0.60 to 3.02 +/- 0.73 pg/mL), while nitric oxide (NO) and forearm blood flow increased by 29% and 14%, respectively. During saline infusion, ET-1, nitric oxide, and forearm blood flow remained unchanged. There was a significant interaction between the effect of decreasing ET-1 levels and the heparin treatment (F, 4.06; df, 3.30; P < .01). The decrease in ET-1 levels was significantly correlated with the increase in forearm blood flow in test 1 (r = .74; P < .01) but not in test 2. During the heparin/insulin period, ET-1 increased by 25%, returning to fasting values; nitric oxide levels increased by 12%; and forearm blood flow remained unchanged. CONCLUSIONS: The present study showed that it is possible to decrease ET-1 levels by use of low-dose heparin infusion in humans. This effect seems mediated by a simultaneous increase in nitric oxide levels and is completely reversed by a mild increase in insulin concentrations.

Adult↗
PubMed1996

Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells: studies using selective phosphodiesterase inhibitors.

The second messenger cAMP has been implicated in the regulation of mammalian and amphibian oocyte maturation. Although a decrease in intraoocyte levels of cAMP precedes germinal vesicle breakdown (GVBD), the gonadotropin induction of ovulation and oocyte maturation is associated with major increases of cAMP in ovarian follicles. In the mammalian system, isolated oocytes undergo spontaneous maturation in vitro but this process is blocked by treatment with a phosphodiesterase (PDE) inhibitor, IBMX, which increases intraoocyte cAMP levels. In contrast, the same inhibitor, when added to cultured follicles for a brief time, increases follicle cAMP levels, followed by the induction of GVBD. To resolve the paradoxical actions of this PDE inhibitor on the maturation of isolated and follicle-enclosed oocytes, we hypothesized that meiotic maturation requires opposing fluctuations of cAMP levels in the somatic granulosa and germ cells. Such opposing fluctuations may result from selective expression and regulation of PDEs in the somatic and germ cell compartments of the follicle. To test this hypothesis, PDE activity was manipulated in different follicular cells using type-specific inhibitors. The impact of the ensuing changes in cAMP levels in the two compartments was monitored by the induction of GVBD. In isolated oocytes, spontaneous GVBD was blocked by two inhibitors of type 3 PDE (cGMP-inhibited: CGI-PDE), milrinone and cilostamide. In contrast, treatment with an inhibitor for type 4 PDE (cAMP-specific), rolipram, was ineffective. These findings suggest that the oocyte expresses type 3 but not type 4 PDE and that increases in intraoocyte cAMP suppress GVBD. This hypothesis was confirmed by in situ hybridization studies with PDE3 and PDE4 probes. PDE3B mRNA was concentrated in oocytes while PDE4D was mainly expressed in granulosa cells. In cultured follicles, LH treatment induced oocyte maturation but the gonadotropin action was blocked by inhibitors of type 3 but not the type 4 PDE inhibitors. Furthermore, treatment with the type 4, but not the type 3, PDE inhibitor mimics the action of LH and induces oocyte maturation, presumably by increasing cAMP levels in granulosa cells. Our findings indicate that PDE subtypes 4 and 3 are located in follicle somatic and germ cells, respectively. Preferential inhibition of PDE 3 in the oocyte may lead to a delay in oocyte maturation without affecting the cAMP-induced ovulatory process in the somatic cells. Conversely, selective suppression of granulosa cell cAMP-PDE may enhance the gonadotropin induction of ovulation and oocyte maturation. Thus, in addition to the well-recognized differential expression and regulation of adenylate cyclase in the somatic and germ cell compartments of the follicle, we suggest that selective regulation and expression of PDEs may be involved in the regulation of cAMP levels and control of oocyte maturation in the preovulatory mammalian follicle.

Animals↗
PubMed1996

Phosphorylation and activation of a cAMP-specific phosphodiesterase by the cAMP-dependent protein kinase. Involvement of serine 54 in the enzyme activation.

A cAMP-specific phosphodiesterase (PDE4D3) is activated in rat thyroid cells by TSH through a cAMP-dependent phosphorylation (Sette, C., Iona, S., and Conti, M.(1994) J. Biol. Chem. 269, 9245-9252). This short term activation may be involved in the termination of the hormonal stimulation and/or in the induction of desensitization. Here, we have further characterized the protein kinase A (PKA)-dependent phosphorylation of this PDE4D3 variant and identified the phosphorylation site involved in the PDE activation. The PKA-dependent incorporation of phosphate in the partially purified, recombinant rat PDE4D3 followed a time course similar to that of activation. Half-maximal activation of the enzyme was obtained with 0.6 microM ATP and 30 nM of the catalytic subunit of PKA. Phosphorylation altered the Vmax of the PDE without affecting the Km for cAMP. Phosphorylation also modified the Mg2+ requirements and the pattern of inhibition by rolipram. Cyanogen bromide cleavage of the 32P-labeled rat PDE4D3 yielded two or three major phosphopeptide bands, providing a first indication that the enzyme may be phosphorylated at multiple sites in a cell-free system. Site-directed mutagenesis was performed on the serine residues present at the amino terminus of this PDE in the context of preferred motifs for PKA phosphorylation. The PKA-dependent incorporation of 32P was reduced to the largest extent in mutants with both Ser13 --> Ala and Ser54 --> Ala substitutions, confirming the presence of more than one phosphorylation site in rat PDE4D3. While substitution of serine 13 with alanine did not affect the activation by PKA, substitution of Ser54 completely suppressed the kinase activation. Similar conclusions were reached with wild type and mutated PDE4D3 proteins expressed in MA-10 cells, where the endogenous PKA was activated by dibutyryl cAMP. Again, the PDE with the Ser54 --> Ala substitution could not be activated by the endogenous PKA in the intact cell. These findings support the hypothesis that the PDE4D3 variant contains a regulatory domain target for phosphorylation at the amino terminus of the protein and that Ser54 in this domain plays a crucial role in activation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Identification of cyclic AMP-phosphodiesterase variants from the PDE4D gene expressed in human peripheral mononuclear cells.

To determine whether the expression of different PDE4D variants is unique to the rat or conserved through evolution, we have characterized the different PDE4D mRNAs expressed in human peripheral blood mononuclear cells. RT-PCR was performed using primers based on rat sequences and mRNAs from mononuclear cells. The specifically amplified fragments had a size identical to that predicted for rat PDE4D1, PDE4D2 and PDE4D3. Sequencing confirmed that these fragments are derived from the human PDE4D gene. Their sequence was highly homologous to that reported for the rat variants. cDNAs corresponding to the entire ORF of human PDE4D2 and PDE4D3 were expressed in mammalian cells, causing a large increase in PDE activity. Western blot analysis of human peripheral blood mononuclear cell extracts demonstrated the presence of proteins corresponding to the recombinant PDE4D1 and PDE4D2. The pattern of splicing and different promoter usage of the PDE4D gene is therefore conserved during evolution, which indicates an important physiological role.

3',5'-Cyclic-AMP Phosphodiesterases↗